This invention generally relates to novel compositions and methods for the treatment of certain cancers. Additionally, this invention relates to novel compositions and methods to screen drugs useful for the treatment of certain cancers.
Cancer is a worldwide problem. The American Cancer Society estimates that over one half million people will die from cancer in the United States alone in 1999. As such, finding novel compositions and methods for the treatment of cancer is of vital interest. The treatment of cancer falls into three general categories: chemotherapy, radiation therapy and surgery. Often, therapies are combined since a combination of therapies often increases the probability the cancer will be eradicated as compared to treatment strategies utilizing a single therapy. Most typically, the surgical excision of large tumor masses is followed by chemotherapy and/or radiation therapy.
Chemotherapeutic agents can work in a number of ways. For example, chemotherapeutic can work by interfering with cell cycle progression or by generating DNA strand breaks. If the cancer cell is not able to overcome the cell cycle blockage or cell injury caused by the therapeutic compound, the cell will often die via apoptotic mechanisms. The use of a single chemotherapeutic agent in the treatment of cancer, with or without surgery or radiation, has several disadvantages. First, the cells may develop resistance to the chemotherapeutic agent. Such resistance results either in the requirement for higher dosages of the drug and/or the renewed spread of the cancer. Chemotherapeutic agents can be toxic to the patient. Therefore, there is a practical upper limit to the amount that a patient can receive. However, if two chemotherapeutic agents are used in concert, the dosage of any single drug can be lowered. This is beneficial to the patient since using lower levels of chemotherapeutic agents is generally safer for the patient. Additionally, cancer cells are less likely to generate resistance to the combination of drugs as they are to a single drug.
The design of drug combinations for the chemotherapeutic treatment of cancer should be approached with the goals of 1) finding a combination that is synergistic with and not merely additive to the first compound with respect to the elimination of the tumor, and 2) finding a second drug that does not potentiate the toxic effects of the first chemotherapeutic agent. These conditions require a great deal of empirical testing of agents known to have anticancer properties with agents that either may have anticancer properties, or that may augment the first agent in other ways. TMZ is currently employed in chemotherapeutic treatment of certain tumors. It works by dramatically increasing the mutation rate of cells undergoing DNA replication. Such cells, because of the high number of mutations which they have acquired as a result of the treatment with TMZ, are rapidly removed by apoptosis, thereby potentially eliminating the tumor. Some tumor cells are resistant to treatment by TMZ due to deficiencies in the mismatch repair (MMR) system in the cell. A defective MMR system prevents the cell from recognizing O6mG DNA adducts thereby making the cell resistant to elimination.
Baer et al., in U.S. Pat. No. 5;731,304, note that the toxicity of temozolomide can be potentiated by agents that inhibit the enzyme O6-alkylguanine DNA alkyltrarisferase (ATase). In particular, they note that O6-benzylguanine (BG) can enhance the toxicity of temozolomide in certain cell lines that exhibit high levels of ATase (e.g. 300-fold in MAWI cells). However, in other cell lines that exhibit lower levels of ATase (e.g. U373 cells) the effect is significantly less.
Mitchell and Dolan (Cancer Chemother Pharmocol 32:59-63, 1993) note that temozolomide (TMZ) and an analogue, 5-(dimethyltriazeno)imidazole-4-caroxamide (DITC), can be effective in enhancing the anti-tumor effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). TMZ and DITC work by depleting cells or tumors of O6-alkylguanine-DNA alkytransferase (AGT). AGT is a DNA repair protein that selectively removes adducts from the O6 position of guanine in DNA by a stoichiometric transfer of the alkyl group to a cysteine moiety. Removal of the alkyl group from the DNA by methylation of the O6 position of the guanine effectively inactivates the AGT. As with the patent referred to above, the disclosed method is limited to specific cells or cancers.
Therefore, what is need is the development of novel therapies that utilize the synergistic properties of two or more therapeutic agents for the treatment of cancer that have a broader range of targets or a different range of targets than those combination therapies already known.
The invention contemplates that temozolomide (TMZ) and methoxyamine (MX) shall be used as a treatment for certain tumors that are resistant to treatment by TMZ alone. Additionally, the invention contemplates the TMZ and inhibitors of poly (ADP-ribose polymerase (PARP) shall be used as a treatment for certain tumors that. are resistant to treatment by TMZ alone. The present invention generally comprises novel compositions to 1) screen for compounds that can potentiate or modulate the therapeutic effect of temozolomide (TMZ) alone or combined with methoxyamine (MX), 2) provide model systems for the study of cancer treatments by agents that modulate DNA repair mechanisms and 3) provide treatments for certain cancers.
With regard to the treatment of cancer, the present invention contemplates methods of treating cancer that utilize TMZ in conjunction with another agent that is capable of potentiating the toxic effect of the TMZ. More specifically, the invention relates to potentiating the effect of TMZ with agents that modify DNA adducts created by TMZ. Even more specifically, the invention relates to potentiating the effect of TMZ with agents that modify N7-methylguanine (N7mG) and N3-methyladenine (N3mA) DNA adducts. One example of such an agent is MX. The present invention is not limited by the order in which the agents are administered. In one embodiment, the agents are administered sequentially. In another embodiment, the agents are administered as a combined formulation (i.e., a formulation comprising methoxyamine and temezolomide). In one embodiment, the present invention contemplates a method of treating cancer comprising: a) providing i) a patient diagnosed with cancer, ii), a first formulation comprising methoxyamine and iii) a second formulation comprising temozolomide; b) administering said first formulation to said patient; and c) administering said second formulation to said patient.
Additionally, the invention relates to potentiating temozolomide with agents that interfere with the ability of PARP to be effective in DNA repair by either competing with PARP, inhibiting PARP or degrading PARP. The present invention is not limited to the agent used to. inhibit PARP activity. In one embodiment, PD128763 is used. In another embodiment 6-AN is used. In another embodiment, 3-AB is used. The present invention is not limited by the order in which the agents are administered. In one embodiment the agents are administered sequentially. In another embodiment, the agents are administered as a combined formulation. In one embodiment, the present invention contemplates a method of treating cancer comprising: a) providing i) a patient diagnosed with cancer, ii) a first formulation comprising a poly-(ADP-ribose)-polymerase inhibitor and iii) a second formulation comprising temozolomide; b) administering said first formulation to said patient; and c) administering said second formulation to said patient.
The present invention is not limited to the method of administration of the treatment. In one embodiment, the treatment is administered orally. In another embodiment the treatment is administered intravenously. In yet another embodiment, the treatment is administered intraperitoneally. In yet another embodiment, the treatment is administered directly to the tumor by injection or, in the case of skin tumors, for example, by direct application of creams or ointments.
As noted above, the present invention also contemplates screening assays to identify drugs that augment the ability of temozolomide to inhibit tumor growth, drugs that augment the ability of temozolomide with methoxyamine to inhibit tumor growth, and drugs that augment the ability of temozolomide with PARP inhibitors to inhibit tumor growth. A variety of assay formats are contemplated for testing the potential of compounds suspected of augmenting the anti-tumor effect of TMZ. In one embodiment, cells are pretreated with the compound suspected of augmenting the anti-tumor effect of temozolomide, followed by treatment with temozolomide. Cell growth and/or cell death are then measured to determine if there is a an anti-tumor effect. In another embodiment, cells are treated with the compound suspected of augmenting the anti-tumor effect of temozolomide at approximately the same time as they are treated with temozolomide. Cell growth and/or cell death are then measured to determine if the regime had an anti-tumor effect. The invention is not limited to any particular measurement technique for apoptosis or cell growth. Various methods are envisioned. For example, mitosiscan be measured by use of fluorescent dyes that intercalate into DNA, by the measurement of H3-thymidine incorporation or by colormetric assays. Such assays permit the use of high throughput screening methods. Apoptosis can be measured by the use of fluorescent dyes that intercalate into DNA, annexin-V staining of phosphotidyl serine residues of the cell surface or morphological changes in cell appearance.
The present invention contemplates a method of screening for compounds that augment the anti-tumor effect of temolozomide comprising: a) providing i) tumor cells, ii) a first formulation comprising a compound suspected of potentiating temolozomide and iii) a second formulation comprising temozolomide; b) contacting said cells with said first formulation; c) contacting said cells with said second formulation; and d) measuring the growth and death rates of said cells.
The present invention contemplates a method of screening for compounds that augment the anti-tumor effect of temolozomide comprising: a) providing i) an experimental animal and ii) tumor cells; iii) a first formulation comprising a compound suspected of potentiating temolozomide and iv) a second formulation comprising temozolomide; b) contacting said tumor cells with said experimental animal in such a way so that the tumor cells will grow in said animal so to make an animal with cancer; c) administering said first formulation to said animal with cancer; d) administering said second formulation to said animal with cancer; and e) measuring the growth and death rates of said cancer cells.
It is not intended that the present invention be limited by the nature of the compounds to be screened in the screening assay of the present invention. For example, a variety of compounds including peptides, organic compounds and nonorganic compounds, are contemplated. Additionally, combinations of compounds are contemplated by the present invention.